Enzymatic detoxification of organophosphorus compounds

ABSTRACT

Compositions and methods are provided for enzymatic detoxification of organophosphorus compounds. The compositions contain a recombinant enzyme expressed by a cloned gene encoding an organophosphorus acid anhydrolase (OPAA-2) from the Alteromonas sp. bacteria, strain JD6.5. These compositions may be prepared as a dry powder and reconstituted with various water sources when needed. The methods involve application of compositions of recombinant OPAA-2 to surfaces or substances contaminated with organophosphorus compounds under conditions which maximize activity and stability of the enzyme.

GOVERNMENTAL INTEREST

The invention described herein may be manufactured, used and licensed by or for the U.S. Government.

This application is a nonprovisional continuation of provisional application Ser. No. 06/015,966, filed on Apr. 16, 1996.

FIELD OF THE INVENTION

The present invention relates generally to the hydrolysis of organophosphorus compounds. More specifically, the present invention relates to the expression of a recombinant bacterial enzyme which is useful for detoxifying cholinesterase-inhibiting organophosphorus compounds such as pesticides and chemical nerve agents and the decontamination of substances contaminated with these compounds.

BACKGROUND OF THE INVENTION

A number of organophosphorus (OP) compounds used by the agriculture industry and the military are highly toxic and thus hazardous to human health and harmful to the environment. For example, acetylcholinesterase-inhibiting OP compounds comprise the active ingredient of pesticides such as paraoxon as well as G-type nerve agents such as Sarin and Soman, etc., developed for chemical warfare. Thus, it is very important to be able to detoxify such OP compounds and to decontaminate surfaces and substances contaminated with these compounds.

Currently, the U.S. Army uses a nerve agent decontamination solution, DS2,which is composed (by weight) of 2% NaOH, 28% ethylene glycol monomethyl ether, and 70% diethylenetriamine (Richardson, G. A. "Development of a package decontamination system", EACR-1 310-17, U.S. Army Edgewood Arsenal Contract Report (1972), herein incorporated by reference). Although this decontamination solution is effective against nerve agents, it is quite toxic, flammable, corrosive, and can threaten the environment. Thus, there is a need for an alternative decontamination technology that is both effective and non hazardous to personnel, equipment, and/or the environment.

One approach being investigated as a potential solution to this problem is enzyme-mediated decontamination. For example, a class of enzymes known as organophosphorus acid (OPA) anhydrolases (OPAA) (EC 3.1.8.2) can catalyze the hydrolysis of a variety of OP compounds, including pesticides and fluorinated "G-type" nerve agents. (See, e.g., Landis, W. G., et al., "Discovery of multiple organofluorophosphate hydrolyzing activities in the protozoan Tetrahymena thermophila", J. Appl. Toxicol., Vol. 7, pp. 35-41, 1987; DeFrank, J. J., et al., "Screening of halophilic bacteria and alteromonas species for organophosphorus hydrolyzing enzyme activity", Chem. Biol. Interact., Vol. 87, pp. 141-148, 1993; each of which is incorporated herein by reference.) Although OPA anhydrolases are produced by both prokaryotic and eukaryotic organisms, their use for enzymatic decontamination requires an abundant supply that is readily and cheaply obtained. Thus, a recombinant microorganism that is easily grown and capable of overexpressing an OPAA having high activity against cholinesterase-inhibiting OP compounds under a variety of conditions would be very desirable.

The first OP-hydrolyzing enzyme gene to be cloned, sequenced, and expressed was that for organophosphorus hydrolase (OPH) (EC 3.1.8.1), which is encoded by the opd gene of Pseudomonas diminuta MG and the Flavobacterium sp. strain ATCC 27551. (See, e.g., McDaniel, C. S. et al., "Cloning and sequencing of a plasmid-borne gene (opd) encoding a phosphotriesterase", J. Bacteriol., Vol. 170:, pp. 2306-2311, 1989, incorporated herein by reference.) The open reading frame of the opd gene contains 975 bases which encode a polypeptide of 325 amino acid residues with a molecular mass of 35 kDa. (See Mulbry, W. and Karns, J., "Parathion hydrolase gene specified by the Flavobacterium opd gene: relationship between the gene and protein", J. Bacteriol., Vol. 171, pp. 6740-6746, 1989, incorporated herein by reference.)

The inventors have recently purified another OPAA with very high OP-hydrolyzing activity from a bacteria strain, Alteromonas sp. strain JD6.5, isolated from Grantsville Warm Springs in Salt Lake City, Ut. (DeFrank J. J. and T.-C. Cheng, "Purification and properties of an organophosphorus acid anhydrolase from a halophilic bacterial isolate", J. of Bacteriol., Vol. 173, pp. 1938-1943, 1991, herein incorporated by reference.) This enzyme, OPAA-2, displays high catalytic activity at a wide pH range (6.0 to 9.5) and temperature range (10° to 65° C.), with optimum activity at pH 8.5 and 50° C. OPAA-2 has hydrolytic activity against a number of OP compounds, including the chemical nerve agents Sarin (GB; o-isopropyl methylphosphonofluoridate), Soman (GD; o-pinacolyimethylphosphonofluoridate), and GF (o-cyclohexyl methylphosphonofluoridate), the nerve-agent-analog diisopropyl fluorophosphate (DFP), and pesticides such as paraoxon.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a system for enzymatic degradation of organophosphorus compounds that is simple and safe for users, equipment, and/or the environment.

It is another object of the present invention to provide a composition for enzymatic degradation of organophosphorus compounds that is compatible with a variety of foams, e.g., fire fighting foams, wetting agents, and degreasers.

These and other objects are satisfied by the present invention which concerns the cloning, sequencing and expression of the gene encoding OPAA-2 from Alteromonas sp. strain JD6.5.

Accordingly, the present invention includes an isolated and purified DNA fragment comprising a DNA sequence encoding a polypeptide having the biological activity of OPAA-2.

The invention also includes a recombinant DNA (rDNA) construct for expressing OPAA-2 in prokaryotic cells. The rDNA construct comprises a prokaryotic promoter operatively linked to a DNA sequence coding for a polypeptide having the biological activity of OPAA-2.

Another aspect of the present invention includes recombinant OPAA-2 (rOPAA-2) formulated for a long shelf life. In particular, the present invention provides rOPAA-2 as a dehydrated powder that can be reconstituted with various water sources.

Yet another aspect of the present invention is the use of rOPAA-2 to detoxify stocks of cholinesterase-inhibiting organophosphorus compounds, to decontaminate surfaces contaminated with these compounds, to remediate contaminated soils, and to treat liquid and solid wastes generated from producing these compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the OPA anhydrolase activity of purified rOPAA-2 against substrates DFP, GB (Sarin), GD (Soman), GF (o-cyclohexyl methylphosphonofluoridate) and NP-GD (chromogenic soman analog). FIG. 1A is a graph of the Km values and FIG. 1B is a graph of the Vmax values.

FIG. 2 shows the effect of varying the ammonium carbonate, (NH₄)₂ CO₃, concentration on the specific activity of rOPAA-2.

FIG. 3 shows the effect of varying the manganese chloride, MnCl₂, concentration on the specific activity of rOPAA-2 buffered in 50 mM (NH₄)₂ CO₃.

FIG. 4 shows the stability of rOPAA-2 in (NH₄)₂ CO₃ buffer at 25° C. and 37° C.

FIG. 5 shows the effect on the specific activity of rOPAA-2 against DFP when lyophilized enzyme is reconstituted with different (NH₄)₂ CO₃ -buffered water sources containing no foam, 6% blast containment foam (AFC-300), or 6% fire-fighting foam (FFF). FIG. 5A is a bar graph of OPAA-2 activity at 0 min. and FIG. 5B is a bar graph of OPAA-2 activity at 60 min. after reconstitution, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the cloning and expression of the gene coding for OPAA-2 from Alteromonas sp. strain JD6.5, also referred to as opaA. To accomplish this, the following experiments were performed.

EXAMPLE 1 CLONING AND SEQUENCING OF opaA Cultivation of Alteromonas Sp. Strain JD6. 5

The A. sp. JD6.5 strain was grown in 10 ml of a medium consisting of the following (per liter): NaCl, 50 g; MgSO₄ --H₂ O, 10 g; Proteose peptone, 10 g; yeast extract, 6 g; and N-2-hydroxyethylpeperazine-N'-2-ethanesulfonic acid (HEPES), 2.5 g (pH 6.8). The 10 ml culture was incubated at 30-37° C., on a rotary shaker at 240 rpm, for 18 to 24 hours. The bacteria cells were then harvested by centrifugation (7,500×g) for 10 min, resuspended in 10 ml of phosphate-buffered saline (PBS) and then collected by centrifugation at 1,000×g for 5 min.

Construction of a Genomic Library Expressing OPAA-2

High molecular weight genomic DNA from the above cultured cells of A. JD6.5 was prepared as described by Davis et al., Basic Methods in Molecular Biology, p. 44-46, Elsevier, N.Y., 1986, herein incorporated by reference. The genomic DNA was partially digested with EcoRI and the resulting 4-8 kb DNA fragments were size fractionated by agarose gel electrophoresis. The separated fragments were purified from the gel with the Geneclean™ II kit (Bio-101, La Jolla, Calif.). The purified DNA fragments were then ligated to EcoRI-digested, phosphatase-treated Lambda ZAP® arms obtained from Stratagene® (La Jolla, Calif.) and then packaged with Gigapack® II Gold packaging extract (Stratagene®). Host strain E. coli XL1-Blue (Stratagene®) was infected with the resulting recombinant phage preparation and plated onto LB agar plates.

Screening of Cloned OPAA-2

To verify that the above genomic library contained OPAA-2 genomic DNA from A. sp. JD6.5, it was screened for the presence of recombinant plaques with an anti-OPAA-2 serum, the preparation of which has been described previously (DeFrank J. J. and T.-C. Cheng, 1991). Approximately 6×10⁴ plaque-forming units (pfu) were plated at a density of 1.5×10⁴ pfu per 90-mm-diameter plate on E. coli XL1-Blue cells. After 6 hours of growth at 37° C., the plates were overlaid with nitrocellulose filters saturated with 10 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and incubated for an additional 3 h. After cooling at 4° C. for 1 h., the filters were then removed from the plates and blocked overnight at room temperature with TBS (50 mM Tris, pH 7.5, 150 mM N2Cl) containing 5% dry skim milk.

Immunodetection of OPAA-2 was carried out by first incubating the filters with a 1:2000 (v/v) dilution of anti-OPAA-2 serum in TBS for 1 h. After three washes in TBS, the filters were incubated with goat anti-rat IgG-alkaline phosphatase conjugate (Fisher Scientific, Pittsburgh, Pa.). Following 3 washes with TBS, the bound alkaline phosphatase was assayed with the substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT). Of 6×10⁴ plaques examined, 5 clones gave strong signals with the anti-OPAA-2 serum.

Excision of Plasmid from the Lambda ZAP® Vector and Subcloning of the Cloned Gene

The excision of plasmid from the positive clones was carried out as described by Short, J. M., et al., "λZAP: a bacteriophage λ expression vector with in vivo excision properties", Nucleic Acids Res., Vol. 16, pp. 7583-7589 (1988), incorporated herein by reference. The recovered plasmids all contained a 6.1 kb EcoRI DNA fragment.

One positive clone, designated pTC651, was chosen for subcloning. This plasmid was digested with PstI and then partially digested with HindIII. The purified PstI-HindIII fragments were subcloned into the PstI and HindIII sites of pBluescript® and the resulting recombinant plasmids used to transform Epicurian Coli® XL1-blue host cells using standard procedures (see e.g., Sambrook, J., et al., Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring harbor, N.Y., 1989, incorporated herein by reference).

The resulting transformants were analyzed for the expression of functional rOPAA-2 by Western analysis and DFP-hydrolyzing activity as described by Cheng, T.-C., et al., "Cloning, Expression and Nucleotide Sequence of a Bacterial Enzyme for Decontamination of Organophosphorus Nerve Agents", Applied and Environmental Microbiology, Vol. 62, pp. 1636-1641 (1996), incorporated herein by reference. The subclone, designated pTC6513, was found to contain the PstI-HindIII fragment, 1.74 kb in length, which encoded functional OPAA-2. Recombinant bacteria cells pTC6513/XL1-blue were deposited on Dec. 18, 1996 with the ATCC (12301 Parklawn Drive, Rockville, Md. 10852) under Accession No. 98281.

DNA Sequencing of the OPA Anhydrolase Gene

The nucleotide sequence of the PstI-HindIII fragment carried by pTC6513 was determined from both strands using the Taq cycle sequencing method and fluorescently-labeled T7 or T3 primers or, when sequence-specific primers were used, fluorescently-labeled dideoxynucleotides. The sequencing reactions were performed by an automated DNA sequencer, Model 373A (Perkin Elmer, Applied Biosystems Div.; Foster City, Calif.), following the manufacturer's protocol. Computer analysis of the obtained DNA sequence was performed with the MacVector® program, version 4.1.4 (Kodak; New Haven, Conn.). The Genbank accession number of the OPAA-2 gene sequence is U29240.

The 1,740 base pair (bp) nucleotide sequence oriented from the PstI site to the HindIII site is shown in SEQ ID NO.: 1. Within this sequence, only one large open reading frame was found. This open reading frame, beginning at nucleotide 36 and terminating at nucleotide 1586, consisted of a 1,551 bp nucleotide sequence encoding a polypeptide of 517 amino acid residues having the deduced amino acid sequence shown in SEQ ID NO.: 2. The calculated molecular weight of this polypeptide is 59 kDa, which is very close to the 60 kDa molecular weight of native OPAA-2 determined by Western analysis.

As is well-known in the art, conservative changes may be made to either or both of the DNA and amino acid sequences of SEQ ID NO.:1 and SEQ ID NO.:2 without significantly affecting the biological activity of the expressed polypeptide. Accordingly, the present invention includes all DNA fragments having a DNA sequence which codes for a polypeptide having the biological activity of OPAA-2. These DNA fragments are identified as those that contain DNA sequences which are substantially homologous to the DNA sequence of SEQ ID NO.:1 or its complementary sequence.

Substantially homologous DNA sequences are defined as having at least about 85% homology over the defined length of the DNA sequences, with at least about 90% homology being preferred and at least about 95% homology being most preferred. Sequences that are substantially homologous may be identified in a Southern hybridization experiment under stringent conditions. Defining stringent conditions for a particular hybridization experiment is within the skill of the art. Generally, hybridization under stringent conditions is performed at about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH (T. Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory (1982), pp. 387-389, incorporated herein by reference). Typical stringent conditions include hybridization in 4×SSC at 65° C. or in 4×SSC, 50% formamide at 42° C., followed by washing in 0.1×SSC at 65° for 30 min.

In addition, it is also well known in the art that a variety of prokaryotic promoters and host microorganisms are capable of expressing recombinant proteins. Accordingly, the present invention is not limited to the use of any particular combination of expression vector and host cell for expression of a DNA fragment comprising a DNA sequence coding for OPAA-2. For example, the following expression systems have been used to overexpress and purify rOPAA-2: pRSET Xpress® Kit (Cat. #K880-01, Invitrogen, San Diego, Calif.); pProEX HT® Protein Expression System (Cat. # 10711-018, Gibco BRL Life Technology, Gaithersburg, Md.); QIAexpress® Type VI Kit (Cat. # 32149, Qiagen, Inc., Chatsworth, Calif.); IMPACT® I System (Cat. # 6500, New England Biolabs, Inc., Beverly, Mass.).

EXAMPLE 2 PURIFICATION OF RECOMBINANT OPAA -2 Purification Procedure

A one liter culture of pTC6513/XL1-blue cells was grown to lag phase, harvested and then frozen. The frozen cells were resuspended in 10 BM buffer (10 mM Bis-Tris propane, 0.1 mM MnCl₂, pH 7.2) at a ratio of 3 ml of buffer for each gram of wet cells. The cells were sonicated for 2 minutes at 4° C. and the cellular debris removed by centrifugation. Approximately 200 mg of protein from the crude lysate was then used to prepare purified rOPAA-2.

To remove nucleic acids, four volumes of BPA1000 (TOSOHAAS, Montgomeryville, Pa.) was added to the lysate and the mixture was incubated at 4° C. for 10 min. The mixture was then centrifuged at 2,000×g for 5 min., resulting in a cell-free extract containing OPAA activity. Further purification of the enzyme through (NH₄)₂ SO₄ fractionation, DEAE ion exchange chromatography and hydroxyapatite chromatography (HA-Ultrogel) was then followed by procedures similar to those described previously. (See Cheng et al., 1996 and DeFrank and Cheng, 1991.) The OPA anhydrolase activity of rOPAA-2 after each step of the purification scheme was determined using the following assay.

Assay for Enzyme Activity

OPAA activity for diisopropyl fluorophosphate (DFP) was assayed by monitoring released fluorine ion concentration with an ion-specific electrode (see Cheng et al., 1996 and DeFrank and Cheng, 1991). The reaction medium contained 3.0 mM DFP, BTP (500 mM NaCl, 50 mM Bis-tris propane, pH 7.2), 0.1 mM MnCl₂, and 2 to 25 μl of enzyme sample in a total volume of 2.5 ml. One unit of OPA anhydrolase activity catalyzes the release of 1.0 μmole of fluorine ion per min. at 25° C.

The specific activity of enzyme recovered after each purification step was calculated (μmole of F- released/min/mg of protein). Protein concentrations were determined calorimetrically using a Coomassie blue assay (Pierce Co., Rockford, Ill.) with bovine serum albumin (BSA) as the standard.

As summarized in Table 1 below, this purification procedure provided an increase in specific activity of the rOPAA-2 enzyme of almost 20 fold, with a 9% recovery. The expressed enzyme constituted about 5% of the total cellular protein in the recombinant cells.

                  TABLE 1     ______________________________________     Purfication of OPA Anhydrolase-2 from Recombinant Cells.               Total   Total   Total Specific               Volume  Protein Activity                                     Activity                                           Purifi-     Purification Step               (ml)    (mg)    (U)   (U/mg)                                           cation                                                 Yield     ______________________________________     Crude Extract               15.0    204.0   3060  15          100     BPA-1000  60.0    144.0   3070  21    1.4   100     (NH.sub.4).sub.2 SO.sub.4               5.2     58.2    3250  56    3.7   106     (45-65%)     DEAE-Sephacel               6.0     7.3     770   105   7.0   25     HA-Ultrogel               1.9     0.9     270   300   20.0  9     ______________________________________      *Protein and activity values are given as mean of triplicate for each of      enzyme preparation.

EXAMPLE 3 SUBSTRATE SPECIFICITY OF rOPAA-2

To evaluate the ability of rOPAA-2 to hydrolyze different OP compounds, the activity of the purified enzyme for DFP, GB, GD, GF and a chromogenic soman analog (NP-GD) was compared. OPA anhydrolase activity was assayed essentially as described above, with the exception that the hydrolysis of NP-GD was determined by monitoring the absorbance at 405 nm for p-nitrophenol (extinction coefficient, 10,101 at pH 7.2). For all OP compounds tested, 0.1 to 3 mM of the OP substrate and 0.3 to 0.4 units of purified rOPAA-2 was used per 2.5 ml reaction.

The kinetic parameters for these OP compounds were determined by a progress curve fit with the Michaelis-Menten equation, using at least five different substrate concentrations per curve. The substrate concentrations used were between approximately 0.3 and 3 times the K_(m). Other concentrations were used in initial range-finding experiments but were not included in the final calculations. Curve fitting was accomplished with the EZ-FIT software which uses the Nelder-Mead simplex and Marquardt nonlinear regression algorithms sequentially (Perella, F. W., "EZ-FIT: a practical curve-fitting microcomputer program for the analysis of enzyme kinetic data on IBM-PC compatible computers", Anal. Biochem., Vol. 174, pp. 437-447, 1980, incorporated herein by reference).

As shown in FIG. 1, the apparent K_(m) values for DFP, GB, GD, GF and the soman analog (NP-GD) were 2.99, 1.57, 2.48, 0.63 and 1.27 mM, respectively. Apparent V_(max) values against these substrates were 230, 442, 151, 652, and 52 μmole/min/mg, respectively. The recombinant enzyme also hydrolyzes paraoxon at about 2% of the rate of DFP (V_(max) : 6.11 μmole/min/mg). Thus, the purified recombinant OPAA-2 is capable of hydrolyzing DFP and a wide variety of chemical nerve agents under standard assay conditions.

To investigate the usefulness of rOPAA-2 for the detoxification of organophosphorus compounds under a variety of conditions that may be present under actual use conditions, the following experiments were performed.

EXAMPLE 4 THE EFFECT OF REACTION CONDITIONS ON rOPAA-2 ACTIVITY

The activity of purified rOPAA-2 for DFP was assayed under various reaction conditions, including different buffers, pH values, and temperatures. The results are shown in Table 2 below.

                  TABLE 2     ______________________________________     Specific activity of rOPAA-2 With Various Assay Conditions.                            Specific Activity     Assay Condition.sup.a  (μmol/min/mg)     ______________________________________     BTP, pH 7.2 (standard condition)                            310.0 ± 11.7     BTP, pH 7.2 + DTT.sup.b                            593.9 ± 36.4     BTP, pH 7.2 + DTT.sup.b  + BSA.sup.c                            309.8 ± 17.6     BTP, pH 7.2 + DTT.sup.b  + glycerol.sup.d                            325.8 ± 20.1     BTP, pH 8.5 + DTT.sup.b                            713.4 ± 31.0     BTP, pH 8.5 + DTT.sup.b  , 55° C.                            842.6 ± 7.1     Na-acetate/NaOH, pH 8.5                            398.7 ± 16.0     NH.sub.4 -acetate, pH 6.80                            451.6 ± 10.2     NH.sub.4 -formate, pH 6.35                            300.5 ± 19.8     NH.sub.4 -chloride, pH 5.85                            119.7 ± 3.1     NH.sub.4 -acetate/NaOH, pH 8.50                            2,405.7 ± 37.3     NH.sub.4 -formate/NaOH, pH 8.50                            2,373.5 ± 37.1     NH.sub.4 -chloride/NaOH, pH 8.50                            2,356.3 ± 113     (NH.sub.4).sub.2 -carbonate/HC1, pH 8.50                            2,064.7 ± 18.2     (NH.sub.4).sub.2 -carbonate, pH 8.70                            1,675.8 ± 23.2     ______________________________________      .sup.a Assays performed as described in Example 2: all buffers at 50 mM      final concentration; temperature at 25° C. unless indicated      otherwise.      .sup.b DTT, 0.1 mM      .sup.c BSA, 0.5 mg/ml      .sup.d Glycerol, 50%

This experiment demonstrated that the specific activity of rOPAA-2 is affected by the assay conditions. For example, the addition of 0.1 mM DTT (dithiothreitol) to the standard assay buffer resulted in an approximately two-fold increase in specific activity of rOPAA-2 at pH 7.2 (compare rows 1 and 2). When the temperature and pH were raised to 55° C. and 8.5 respectively, the specific activity of rOPAA-2 in the presence of DTT increased about 2.5-fold (compare rows 1 and 6). However, the most striking increases in specific activity occurred when the reaction buffer was changed from BTP to an ammonium-based buffer and the pH adjusted to pH 8.5 or greater (ammonium carbonate dissolved in distilled water has a pH value of around 8.6-8.7), with increases between 5- and 8- fold (compare row 1 with rows 11-15). These results suggest that rOPAA-2 is stimulated by the ammonium ion.

Since the rOPAA-2 showed a high level of anhydrolase activity when buffered with environmentally safe and inexpensive (NH₄)₂ CO₃, the effect of varying the concentration of (NH₄)₂ CO₃ was then studied. In this experiment, the reaction medium contained between 0 and 250 mM (NH₄)₂ CO₃, 3.0 mM DFP, 0.1 mM MnCl₂, and 0.3-0.4 units of rOPAA-2 in a total volume of 2.5 ml. As shown in FIG. 2, rOPAA-2 exhibited a high level of OPA anhydrolase activity over a broad range of (NH₄)₂ CO₃ concentrations, with activity being greatest at 50 mM (NH₄)₂ CO₃.

The activity of rOPAA-2 for DFP in 50 mM (NH₄)₂ CO₃, pH 8.7, with varying MnCl₂ concentrations was also investigated. The results of this experiment, which are shown in FIG. 3, demonstrate that rOPAA-2 is also active over a broad MnCl₂ concentration range, with the greatest OPA anhydrolase activity at ≧0.1 mM MnCl₂.

In addition to having OPA anhydrolase activity, it is desirable that an enzymatic detoxification agent meet several other criteria. For example, the enzyme solution needs to be stable for a time period sufficient to allow its application to the contaminated personnel, equipment or field, and to allow degradation of a significant amount of the contaminating OP compound(s). Also, to reduce the logistical burden of field application, the OPAA enzyme should have a long shelf life when packaged in a lyophilized form and be highly active when reconstituted with whatever water that is readily available when needed. Finally, the enzyme should be compatible with foam or spray systems commonly used in fire-fighting operations, since these systems are uncomplicated and useful for applying the enzyme to large areas. The ability of rOPAA-2 to meet these criteria was assessed in the following examples.

EXAMPLE 5 STABILITY OF rOPAA-2 IN (NH₄)₂ CO₃ BUFFER

Recombinant OPAA-2 was suspended in 50 mM (NH₄)₂ CO₃ (pH 8.7) with or without 0.1 mM MnCl₂ and was incubated at 25° C. or 37° C. up to 120 min. and then assayed for activity against DFP as described in Example 1. The data from this experiment are shown in FIG. 4. The enzyme was more stable in the presence of MnCl₂ at both temperatures and had the highest initial activity at 37° C. in the presence of MnCl₂. However, because the enzyme exhibited a much smaller decrease in activity when incubated in the presence of MnCl₂ for 2 hours at 25° C. than when incubated for 2 hours at 37° C., it had the highest activity by the end of the incubation period. Thus, the stability of enzyme activity in (NH₄)₂ CO₃ -buffered solutions containing 0.05-1.0 mM MnCl₂ at temperatures around 25° C. is sufficient for practical applications. Moreover, (NH₄)₂ CO₃ is an inexpensive and environmentally safe buffer.

EXAMPLE 6 EFFECT OF STORAGE CONDITIONS AND RECONSTITUTION SOLUTIONS ON rOPAA-2 ACTIVITY

The enzyme was lyophilized in the presence or absence of 250 mM trehalose (α-D-glucopyranosyl-α-D-glucopyranoside) with either 5 volumes of a solution containing 50 mM (NH₄)₂ CO₃, pH 8.7 and 0.1 mM MnCl₂ or, as a control, 5 volumes of 10 BM (10 mM Bis-Tris-propane, pH 7.2) containing 0.1 mM MnCl₂ and 0.1 mM DTT (without trehalose). The lyophilized enzyme preparations were stored up to one year at various temperatures between -80° C. and +37° C. and then reconstituted in the presence or absence of 6% foam agent with 0.1 mM MnCl₂ and either BTP or (NH₄)₂ CO₃ buffer (50 mM, pH 8.7) prepared with distilled water, municipal drinking water collected at Baltimore, Md. (approximately 1 mg/liter residual chlorine), or Atlantic Ocean water collected at Ocean City, Md. The foam agent used was either blast containment foam (AFC-3800) (Sandia National Laboratory, Albuquerque, N. Mex.) or fire-fighting foam (FFF) (Ansul Fire Protection Inc., Marinette, Wis.). The various reconstituted rOPAA-2 enzyme solutions were then assayed for OPA anhydrolase activity of DFP at 0 minutes or 60 minutes after reconstitution. There was no foam generation in these experiments.

Enzyme lyophilized with trehalose and (NH₄)₂ C0₃ was found to be stable at -80° C. to +37° C. for up to a year with no apparent loss of activity (data not shown). The importance of trehalose in stabilizing the lyophilized enzyme preparations was demonstrated by the results with rOPAA-2 lyophilized with either (NH₄)₂ CO₃ or 10 BM. Without trehalose, the enzyme lost >90% of its activity.

The effect of the source of the water used to prepare the various (NH₄)₂ CO₃ reconstituting solutions on the activity of rOPAA-2 lyophilized in (NH₄)₂ CO₃ and trehalose is shown in FIGS. 5A and 5B. Lyophilized enzyme reconstituted in the absence of foam with distilled water or drinking water did not differ with respect to OPA anhydrolase activity at either 0 min (FIG. 5A) or 60 min (FIG. 5B) after reconstitution. In contrast, lyophilized enzyme suspended in ocean water containing no foam exhibited less activity. These results demonstrate that rOPAA-2 can be stored as a lyophilized powder in compact containers and reconstituted when needed with (NH₄)₂ CO₃ -buffer distilled water or (NH₄)₂ CO₃ -buffered drinking water to yield a enzyme solution with high OP-hydrolyzing activity.

In the presence of AFC-380, approximately 60% and 45% of the enzyme activity were retained in distilled water and drinking water, respectively, compared with the control with no foam agent. These activities are still higher than untreated enzyme assayed under standard conditions (BTP, pH 7.2 at 25°)(Table 1). In the presence of the other fire fighting foam (FFF) tested, only 30% and 15% of the activity was retained in (NH₄)₂ CO₃ -buffered distilled water and drinking water, respectively. When the lyophilized enzyme was reconstituted in (NH₄)₂ CO₃ -buffered ocean water with or without foam agent, enzyme activities were greatly reduced by about 85-90%.

The effect of the reconstituting solution on the stability of the resulting enzyme solution is shown in FIG. 5B. One hour after reconstitution of the lyophilized enzyme with distilled or drinking water in the absence of foam, the enzyme activity decreased by approximately 25%. Enzyme reconstituted in the presence of either 6% AFC-380 or 6% FFF exhibited a decrease in activity of about 20% in one hour. These results demonstrate that an enzyme solution that is quite stable for at least one hour is produced when rOPAA-2 , lyophilized in 250 mM trehalose, 50 mM (NH₄)₂ CO₃, pH 8.7, and 0.1 mM MnCl₂, is reconstituted with 6% AFC-380 and (NH₄)₂ CO₃ -buffered water (either distilled or drinking water).

In contrast, a foam agent did not moderate the decrease in activity of the enzyme reconstituted with (NH₄)₂ CO₃ -buffered ocean water. Without foam agent, the activity of the enzyme in ocean water decreased by 34% during the one hour period, while the presence of 6% AFC-380 or 6% FFF in the ocean water resulted in a decrease in activity over the same period of 60% and 47%, respectively.

The above results demonstrated the feasibility of a foam-based enzyme system for the detoxification of chemical nerve agents and related organophosphorus compounds. The activity of rOPAA-2 in a number of other foams, wetting agents and degreasers was also examined and is described in the following example.

EXAMPLE 7 ACTIVITY OF rOPAA-2 IN VARIOUS BIODEGRADABLE AND WATER SOLUBLE MATERIALS

In this experiment, rOPAA-2 non-lyophilized was assayed in distilled water containing 50 mM (NH₄)₂ CO₃ and 0.1 mM MnCl₂ in the absence (control) or presence of one of various biodegradable and water soluble materials. The resulting enzyme solutions were assayed at 25° C. for OP-hydrolyzing activity using 3.0 mM DFP as the substrate and 0.3-0.4 units of rOPAA-2 in a total volume of 2.5 ml. The average of the results obtained in 3 assays for each substance are shown in Table 3 below.

                  TABLE 3     ______________________________________     Specific activity of rOPAA-2 in biodegradable and     water soluble materials.                                   Conc.  Sp.                                   Used   Activity     Material      Property        (%)    (U/mg)     ______________________________________     Control       N/A             N/A    1950     AFC-380 ® blast-containment foam                                    6     1050     (Sandia Nat. Lab.,     Albuquerque, NM)     FFF ®     fire fighting foam                                    6     460     (3M, St. Paul, MN)     BioSolve ®                   fire fighting wetting agent                                    6     1030     (Westford Co.,     Westford, MA)     Cold Fire-Retardant ®                   fire retarding agent                                   10     3520     (Firefreeze, Rockaway, NJ)     Cold Fire ®                   fire suppressing agent                                   10     2340     (Firefreeze, Rockaway, NJ)     BV 406LF ®                   degreaser/cleaner                                   10     1430     (Firefreeze, Rockaway, NJ)     Odor Seal ®                   wetting agent/odor                                   10     1980     (Firefreeze, Rockaway, NJ)                   removing     Star Cleaning Miracle ®                   wetting agent/oil removing                                   30     670     (Star, Hudsonville, MI)     SC-1000 ® wetting agent/degreaser                                    5     650     (Gemtek, Phoenix, AZ)     Supersolve ®                   wetting agent/degreaser                                   10     440     (Gemtek, Phoenix, AZ)     Blue Base ®                   degreaser        8     140     (Neutron, Torrance, CA)     ______________________________________

These data indicate that the activity of rOPAA-2 is fairly stable or even enhanced in a number of these water soluble and biodegradable materials, including AFC-380, BioSolve, Cold Fire-Retardant, Cold Fire, BV 406LF, and Odor Seal. Since OP compounds are often oily in nature, the use of these environmentally safe materials in the decontamination system should assist in the solubilization of the chemical agents for enzyme action. Thus, the combination of one or more of these biodegradable materials with a composition comprising rOPAA-2 prepared in (NH₄)₂ CO₃ buffer and preferably also comprising Mn⁺⁺ offers considerable potential as an enzyme-based detoxification system.

In conclusion, the present invention provides a recombinant DNA construct coding for OPAA-2, cells transformed with this construct, rOPAA-2 produced by these transformed cells, and compositions comprising rOPAA-2 which are useful for safe and efficient degradation of chemical nerve agents and other related organophosphorus compounds. While the preceding examples used rOPAA-2 produced by pTC513/XL1-blue cells, the term rOPAA-2 is intended to include polypeptides having the biological activity of OPAA-2 which are produced by other recombinant microorganisms.

Because rOPAA-2 can also catalyze the hydrolysis of pesticides, a composition comprising rOPAA-2 may also be used to decontaminate agricultural equipment, remediate contaminated soils, and treat waste water generated from production facilities. The composition may also comprise MnCl₂ for enhanced stability. In addition, the composition may be used at higher temperatures for higher OPAA-2 activity.

The composition may be incorporated with a foam, wetting agent or degreaser prior to application to aid in the application of the composition and solubilization of organophosphorus compounds. The resulting mixture may be applied to the surface to be decontaminated either manually or with a foam or spray system. Foam and spray systems are uncomplicated and have been used extensively for fire-fighting operations for many years.

Applying the rOPAA-2 composition with foam offers the advantage that the foam would act as a "blanket" to retard evaporation and shield the enzyme from inhibitory environmental elements. In addition, foam could also stabilize the enzyme and thus prolong its activity in the field. Because this foam-based detoxification system would not jeopardize user safety or harm the environment, it could be applied topically to personnel, equipment, agricultural fields or other large areas contaminated with OP compounds.

While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

    __________________________________________________________________________     #             SEQUENCE LISTING     - (1) GENERAL INFORMATION:     -    (iii) NUMBER OF SEQUENCES: 2     - (2) INFORMATION FOR SEQ ID NO:1:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 1740 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: double               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA (genomic)     -    (iii) HYPOTHETICAL: NO     -     (iv) ANTI-SENSE: NO     -     (vi) ORIGINAL SOURCE:     #sp.      (A) ORGANISM: Alteromonas               (B) STRAIN: JD6.5     -    (vii) IMMEDIATE SOURCE:               (B) CLONE: pTC6513     -     (ix) FEATURE:     10.sub.-- - #signalKEY:               (B) LOCATION: 14..18     -     (ix) FEATURE:               (A) NAME/KEY: RBS               (B) LOCATION: 24..30     -     (ix) FEATURE:               (A) NAME/KEY: CDS               (B) LOCATION: 36..1586     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     #TTA GCG GTG        53G CAAAGTGGGT GATAC ATG AAT AAA     #   Met Asn Lys Leu Ala Val     #  5  1     - TTA TAC GCT GAA CAT ATT GCA ACC TTG CAA AA - #G CGC ACG CGC GAA ATT      101     Leu Tyr Ala Glu His Ile Ala Thr Leu Gln Ly - #s Arg Thr Arg Glu Ile     #             20     - ATC GAG CGC GAA AAC CTA GAC GGT GTT GTT TT - #C CAT TCT GGC CAG GCG      149     Ile Glu Arg Glu Asn Leu Asp Gly Val Val Ph - #e His Ser Gly Gln Ala     #         35     - AAG CGC CAG TTC TTA GAC GAT ATG TAC TAC CC - #G TTT AAG GTG AAT CCA      197     Lys Arg Gln Phe Leu Asp Asp Met Tyr Tyr Pr - #o Phe Lys Val Asn Pro     #     50     - CAA TTT AAG GCC TGG TTG CCA GTG ATA GAT AA - #T CCA CAC TGT TGG ATT      245     Gln Phe Lys Ala Trp Leu Pro Val Ile Asp As - #n Pro His Cys Trp Ile     # 70     - GTC GCG AAT GGC ACT GAT AAG CCA AAG TTG AT - #T TTC TAT CGC CCT GTG      293     Val Ala Asn Gly Thr Asp Lys Pro Lys Leu Il - #e Phe Tyr Arg Pro Val     #                 85     - GAC TTT TGG CAC AAG GTC CCC GAT GAG CCG AA - #T GAG TAT TGG GCT GAC      341     Asp Phe Trp His Lys Val Pro Asp Glu Pro As - #n Glu Tyr Trp Ala Asp     #            100     - TAC TTT GAT ATT GAA CTG CTA GTG AAA CCG GA - #T CAG GTA GAA AAG TTA      389     Tyr Phe Asp Ile Glu Leu Leu Val Lys Pro As - #p Gln Val Glu Lys Leu     #       115     - CTA CCC TAT GAT AAG GCG CGA TTT GCA TAT AT - #T GGC GAA TAC TTG GAA      437     Leu Pro Tyr Asp Lys Ala Arg Phe Ala Tyr Il - #e Gly Glu Tyr Leu Glu     #   130     - GTC GCT CAA GCT TTG GGT TTT GAG CTG ATG AA - #T CCG GAG CCG GTA ATG      485     Val Ala Gln Ala Leu Gly Phe Glu Leu Met As - #n Pro Glu Pro Val Met     135                 1 - #40                 1 - #45                 1 -     #50     - AAC TTT TAT CAT TAC CAC CGT GCC TAC AAA AC - #G CAG TAC GAA CTT GCT      533     Asn Phe Tyr His Tyr His Arg Ala Tyr Lys Th - #r Gln Tyr Glu Leu Ala     #               165     - TGT ATG CGT GAG GCG AAT AAA ATC GCT GTA CA - #A GGT CAC AAA GCT GCG      581     Cys Met Arg Glu Ala Asn Lys Ile Ala Val Gl - #n Gly His Lys Ala Ala     #           180     - CGA GAT GCG TTT TTT CAA GGC AAG TCC GAA TT - #T GAA ATT CAA CAA GCC      629     Arg Asp Ala Phe Phe Gln Gly Lys Ser Glu Ph - #e Glu Ile Gln Gln Ala     #       195     - TAC CTG TTA GCG ACC CAA CAC AGC GAA AAT GA - #C AAC GCT TAC GGC AAC      677     Tyr Leu Leu Ala Thr Gln His Ser Glu Asn As - #p Asn Ala Tyr Gly Asn     #   210     - ATT GTG GCG CTA AAT GAA AAC TGC GCC ATT TT - #G CAC TAC ACG CAC TTT      725     Ile Val Ala Leu Asn Glu Asn Cys Ala Ile Le - #u His Tyr Thr His Phe     215                 2 - #20                 2 - #25                 2 -     #30     - GAT CGT GTT GCT CCT GCT ACC CAT CGT TCT TT - #T TTG ATT GAC GCT GGC      773     Asp Arg Val Ala Pro Ala Thr His Arg Ser Ph - #e Leu Ile Asp Ala Gly     #               245     - GCC AAC TTC AAT GGT TAC GCA GCC GAT ATT AC - #T CGA ACC TAT GAC TTT      821     Ala Asn Phe Asn Gly Tyr Ala Ala Asp Ile Th - #r Arg Thr Tyr Asp Phe     #           260     - ACT GGT GAA GGG GAA TTT GCT GAG CTT GTT GC - #C ACC ATG AAG CAG CAC      869     Thr Gly Glu Gly Glu Phe Ala Glu Leu Val Al - #a Thr Met Lys Gln His     #       275     - CAA ATT GCA CTA TGT AAC CAG TTG GCG CCT GG - #C AAG TTA TAT GGT GAG      917     Gln Ile Ala Leu Cys Asn Gln Leu Ala Pro Gl - #y Lys Leu Tyr Gly Glu     #   290     - TTA CAC CTT GAT TGT CAC CAA CGT GTG GCG CA - #A ACA CTG AGT GAC TTT      965     Leu His Leu Asp Cys His Gln Arg Val Ala Gl - #n Thr Leu Ser Asp Phe     295                 3 - #00                 3 - #05                 3 -     #10     - AAC ATC GTC GAC TTA TCG GCC GAT GAG ATT GT - #T GCC AAA GGC ATT ACC     1013     Asn Ile Val Asp Leu Ser Ala Asp Glu Ile Va - #l Ala Lys Gly Ile Thr     #               325     - TCC ACG TTC TTC CCA CAT GGT TTA GGC CAT CA - #T ATT GGT TTA CAA GTA     1061     Ser Thr Phe Phe Pro His Gly Leu Gly His Hi - #s Ile Gly Leu Gln Val     #           340     - CAT GAT GTG GGT GGT TTT ATG GCT GAC GAG CA - #G GGC GCA CAC CAA GAG     1109     His Asp Val Gly Gly Phe Met Ala Asp Glu Gl - #n Gly Ala His Gln Glu     #       355     - CCG CCT GAA GGT CAC CCA TTC CTG CGT TGC AC - #G CGT AAG ATT GAA GCG     1157     Pro Pro Glu Gly His Pro Phe Leu Arg Cys Th - #r Arg Lys Ile Glu Ala     #   370     - AAT CAA GTA TTT ACC ATT GAA CCT GGG TTG TA - #C TTT ATT GAT TCC TTG     1205     Asn Gln Val Phe Thr Ile Glu Pro Gly Leu Ty - #r Phe Ile Asp Ser Leu     375                 3 - #80                 3 - #85                 3 -     #90     - CTC GGT GAT TTA GCA GCG ACA GAT AAT AAT CA - #G CAT ATT AAT TGG GAC     1253     Leu Gly Asp Leu Ala Ala Thr Asp Asn Asn Gl - #n His Ile Asn Trp Asp     #               405     - AAG GTC GCA GAG CTT AAG CCT TTC GGT GGT AT - #T CGT ATT GAG GAC AAT     1301     Lys Val Ala Glu Leu Lys Pro Phe Gly Gly Il - #e Arg Ile Glu Asp Asn     #           420     - ATC ATT GTT CAC GAA GAC AGC CTT GAG AAT AT - #G ACT CGC GAG CTA AGA     1349     Ile Ile Val His Glu Asp Ser Leu Glu Asn Me - #t Thr Arg Glu Leu Arg     #       435     - GCT CGA TTA ACC ACC CAT TCA CTG CGG GGC CT - #A AGT GCT CCG CAG TTT     1397     Ala Arg Leu Thr Thr His Ser Leu Arg Gly Le - #u Ser Ala Pro Gln Phe     #   450     - TCT ATC AAT GAT CCT GCC GTT ATG TCT GAA TA - #C TCA TAC CCT AGT GAG     1445     Ser Ile Asn Asp Pro Ala Val Met Ser Glu Ty - #r Ser Tyr Pro Ser Glu     455                 4 - #60                 4 - #65                 4 -     #70     - CCC TTA AGT TAC GAA GAA GAA ATC AAA AAG AG - #C ACT TTT ATT GTG CAT     1493     Pro Leu Ser Tyr Glu Glu Glu Ile Lys Lys Se - #r Thr Phe Ile Val His     #               485     - GTG CGC ACA CGC CGG ATA TTA GTG CGG CGA AG - #G ACT TTA TCG CCG ATA     1541     Val Arg Thr Arg Arg Ile Leu Val Arg Arg Ar - #g Thr Leu Ser Pro Ile     #           500     - TTA ATC GCC GTT ACC CCG ATG CCC GCC ATA AC - #T GCT GGG CTC ATG     1586     Leu Ile Ala Val Thr Pro Met Pro Ala Ile Th - #r Ala Gly Leu Met     #       515     - TAGCAGGGGC TCCAGGAGGC AGCCATGTGT ATGGCTTTTC CGATGATGGC GA - #GCCCAATG     1646     - GGACTGCTGG CAAACCAATG CTGAATGTGC TCCAAGGCAG TGGCCTTGGC GA - #AATTTGTG     1706     #      1740        CTTC GGTGGTATTA AGCT     - (2) INFORMATION FOR SEQ ID NO:2:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 517 amino               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     - Met Asn Lys Leu Ala Val Leu Tyr Ala Glu Hi - #s Ile Ala Thr Leu Gln     #                 15     - Lys Arg Thr Arg Glu Ile Ile Glu Arg Glu As - #n Leu Asp Gly Val Val     #             30     - Phe His Ser Gly Gln Ala Lys Arg Gln Phe Le - #u Asp Asp Met Tyr Tyr     #         45     - Pro Phe Lys Val Asn Pro Gln Phe Lys Ala Tr - #p Leu Pro Val Ile Asp     #     60     - Asn Pro His Cys Trp Ile Val Ala Asn Gly Th - #r Asp Lys Pro Lys Leu     # 80     - Ile Phe Tyr Arg Pro Val Asp Phe Trp His Ly - #s Val Pro Asp Glu Pro     #                 95     - Asn Glu Tyr Trp Ala Asp Tyr Phe Asp Ile Gl - #u Leu Leu Val Lys Pro     #           110     - Asp Gln Val Glu Lys Leu Leu Pro Tyr Asp Ly - #s Ala Arg Phe Ala Tyr     #       125     - Ile Gly Glu Tyr Leu Glu Val Ala Gln Ala Le - #u Gly Phe Glu Leu Met     #   140     - Asn Pro Glu Pro Val Met Asn Phe Tyr His Ty - #r His Arg Ala Tyr Lys     145                 1 - #50                 1 - #55                 1 -     #60     - Thr Gln Tyr Glu Leu Ala Cys Met Arg Glu Al - #a Asn Lys Ile Ala Val     #               175     - Gln Gly His Lys Ala Ala Arg Asp Ala Phe Ph - #e Gln Gly Lys Ser Glu     #           190     - Phe Glu Ile Gln Gln Ala Tyr Leu Leu Ala Th - #r Gln His Ser Glu Asn     #       205     - Asp Asn Ala Tyr Gly Asn Ile Val Ala Leu As - #n Glu Asn Cys Ala Ile     #   220     - Leu His Tyr Thr His Phe Asp Arg Val Ala Pr - #o Ala Thr His Arg Ser     225                 2 - #30                 2 - #35                 2 -     #40     - Phe Leu Ile Asp Ala Gly Ala Asn Phe Asn Gl - #y Tyr Ala Ala Asp Ile     #               255     - Thr Arg Thr Tyr Asp Phe Thr Gly Glu Gly Gl - #u Phe Ala Glu Leu Val     #           270     - Ala Thr Met Lys Gln His Gln Ile Ala Leu Cy - #s Asn Gln Leu Ala Pro     #       285     - Gly Lys Leu Tyr Gly Glu Leu His Leu Asp Cy - #s His Gln Arg Val Ala     #   300     - Gln Thr Leu Ser Asp Phe Asn Ile Val Asp Le - #u Ser Ala Asp Glu Ile     305                 3 - #10                 3 - #15                 3 -     #20     - Val Ala Lys Gly Ile Thr Ser Thr Phe Phe Pr - #o His Gly Leu Gly His     #               335     - His Ile Gly Leu Gln Val His Asp Val Gly Gl - #y Phe Met Ala Asp Glu     #           350     - Gln Gly Ala His Gln Glu Pro Pro Glu Gly Hi - #s Pro Phe Leu Arg Cys     #       365     - Thr Arg Lys Ile Glu Ala Asn Gln Val Phe Th - #r Ile Glu Pro Gly Leu     #   380     - Tyr Phe Ile Asp Ser Leu Leu Gly Asp Leu Al - #a Ala Thr Asp Asn Asn     385                 3 - #90                 3 - #95                 4 -     #00     - Gln His Ile Asn Trp Asp Lys Val Ala Glu Le - #u Lys Pro Phe Gly Gly     #               415     - Ile Arg Ile Glu Asp Asn Ile Ile Val His Gl - #u Asp Ser Leu Glu Asn     #           430     - Met Thr Arg Glu Leu Arg Ala Arg Leu Thr Th - #r His Ser Leu Arg Gly     #       445     - Leu Ser Ala Pro Gln Phe Ser Ile Asn Asp Pr - #o Ala Val Met Ser Glu     #   460     - Tyr Ser Tyr Pro Ser Glu Pro Leu Ser Tyr Gl - #u Glu Glu Ile Lys Lys     465                 4 - #70                 4 - #75                 4 -     #80     - Ser Thr Phe Ile Val His Val Arg Thr Arg Ar - #g Ile Leu Val Arg Arg     #               495     - Arg Thr Leu Ser Pro Ile Leu Ile Ala Val Th - #r Pro Met Pro Ala Ile     #           510     - Thr Ala Gly Leu Met             515     __________________________________________________________________________ 

We claim:
 1. A purified and isolated DNA fragment, comprising a nucleotide sequence encoding an organophosphorus acid anhydrolase endogenous to Alteromonas species having the biological activity of organophosphorus acid anhydrolase-2.
 2. The DNA fragment of claim 1, wherein said nucleotide sequence hybridizes to SEQ ID NO:1 under stringent conditions.
 3. The DNA fragment of claim 1, wherein said nucleotide sequence comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2.
 4. The DNA fragment of claim 1, wherein said nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:1 or its complementary sequence.
 5. A recombinant DNA construct for expressing an organophosphorus acid anhydrolase endogenous to Alteromonas species in prokaryotic cells, which comprises a prokaryotic promoter operatively linked to a DNA fragment comprising a nucleotide sequence encoding an organophosphorus acid anhydrolase having the biological activity of organophosphorus acid anhydrolase-2.
 6. The recombinant DNA construct of claim 5, wherein said nucleotide sequence hybridizes to SEQ ID NO:1 under stringent conditions.
 7. The recombinant DNA construct of claim 5, wherein said nucleotide sequence encodes the amino acid sequences shown in SEQ ID NO:2.
 8. The recombinant DNA construct of claim 5, wherein said nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:1 or its complementary sequence.
 9. The recombinant DNA construct according to claim 5, wherein said construct is pTC6513.
 10. A recombinant microorganism comprising a host cell transformed by the recombinant DNA construct of claim
 5. 11. A recombinant microorganism comprising a host cell transformed by the recombinant DNA construct of claim
 6. 12. A recombinant microorganism comprising a host cell transformed by the recombinant DNA construct of claim
 7. 13. A recombinant microorganism comprising a host cell transformed by the recombinant DNA construct of claim
 8. 14. A recombinant microorganism comprising a host cell transformed by the recombinant DNA construct of claim
 9. 